﻿A phylogenetic study of Micareamelaeniza and similar-looking species (Pilocarpaceae) unveils hidden diversity and clarifies species boundaries and reproduction modes

﻿Abstract Micarea (Ascomycota, Pilocarpaceae) is a large cosmopolitan genus of crustose lichens. We investigated molecular systematics and taxonomy of the poorly known Micareamelaeniza group focussing on M.melaeniza, M.nigella and M.osloensis. A total of 54 new sequences were generated and using Bayesian and maximum likelihood analysis of two markers (nuITS and mtSSU), we discovered two previously unrecognized phylogenetic lineages, one of which is described here as Micareaeurasiatica Kantelinen & G. Thor, sp. nov., morphologically characterized by pycnidia that are sessile to emergent, cylindrically shaped, with greenish-black K+ olive green, wall pigmentation and containing large mesoconidia up to 6 µm in length. The species is known from Japan and Finland. In addition, we show that the reproduction biology of M.osloensis has been poorly understood and that the species often occurs as an anamorph with stipitate pycnidia. We present a species synopsis and notes on pigments. Our research supports previous results of asexuality being an important reproductive strategy of species growing on dead wood.


Introduction
Species of the genus Micarea Fr. are lichenized ascomycetes belonging to the family Pilocarpaceae.Currently, more than 140 species are known, and new species are continually described (e.g., Czarnota 2007;Czarnota and Guzow-Krzemińska 2010;Sérusiaux et al. 2010;Guzow-Krzemińska et al. 2016, 2019;van den Boom et al. 2017;Kantvilas and Coppins 2019;Launis and Myllys 2019;Launis et al. 2019a, b;van den Boom et al. 2020;Vondrák et al. 2022;Index Fungorum 2023).Micarea species are globally distributed with representatives found on all continents.These species occur across a wide range of habitats and grow on substrates such as bark, dead wood, rocks, soil and bryophytes.Some Micarea species are specialized and prefer specific habitats, such as dead wood in old-growth forests (Fig. 1).Their typical substrate has acidic pH (e.g.Coppins 1983;Czarnota 2007).
Despite the diversity and global presence of Micarea species, they are often overlooked and poorly understood.Several factors contribute to the challenge of identifying them.First, they are typically small.Second, Micarea exhibits a wide range of sexual and asexual propagules, including ascospores, three types of conidia (micro-, meso-, and macroconidia), and thallus fragments called goniocysts which likely serve as asexual propagules including both symbiotic partners.Some Micarea species are primarily sexual, while others often lack sexual structures but form numerous pycnidia where asexual conidia are produced (e.g.Coppins 1983;Czarnota 2007;Kantelinen et al. 2022a).Third, some Micarea species display intraspecific colour variations, which depend on light exposure and corresponding pigment levels.For example, the Sedifolia-grey pigment, commonly found in the apothecia of Micarea species, varies in concentration from light grey in shaded situations to almost black in well-lit habitats (Coppins 1983;Czarnota 2007;Launis et al. 2019aLaunis et al. , 2019b)).Due to these challenges, identifying Micarea species typically requires careful examinations of microscopic features, chemical testing (spot tests and TLC) and/or DNA sequencing.
Significant progress in the understanding of species boundaries and diversity within Micarea has been achieved through the application of molecular methods (Czarnota and Guzow-Krzemińska 2010;Sérusiaux et al 2010;Guzow-Krzemińska et al. 2016;van den Boom et al. 2017;Launis et al. 2019 a, b).However, some infra-generic groups in Micarea have been more studied than others.One group that remains poorly understood and has been sequenced only rarely is Micarea melaeniza Hedl.and similar species, i.e.M. anterior (Nyl.)Hedl., M. botryoides (Nyl.)Coppins, M. deminuta Coppins, M. denigrata (Fr.)Hedl., M. melaeniza, M. misella (Nyl.)Hedl., M. nigella Coppins, M. olivacea Coppins and M. osloensis (Th.Fr.) Hedl..These species share morphological characteristics, such as a thin or endosubstratal thallus, small (0.1-0.3 mm wide) dark apothecia and/or dark stipitate mesopycnidia, as well as often simple to one septate ascospores.Furthermore, M. botryoides, M. deminuta, M. melaeniza, M. nigella, M. olivacea and M. osloensis are similar in having dark hypothecium and dimorphic paraphyses.The Cinereorufa-green (K+ green, HNO 3 + purple) and Superba-brown (K-, HNO 3 -) pigments are often present, as well as sometimes Melaena-red (K+ green, HNO 3 + purple-red).Most of these species are obligate or facultative lignicoles (Coppins 1983, Czarnota 2007).Despite their morphological and ecological similarities, all the species are not necessarily closely related (Coppins 1983;Andersen and Ekman 2005;Sérusiaux et al. 2010).We aim to clarify the molecular systematics and morphology of the poorly known M. melaeniza and its similar-looking species, focusing especially on M. melaeniza, M. nigella and M. osloensis, species that are morphologically challenging to identify.We use phenotypic characters and sequence data from two loci (nuITS and mtSSU).Additionally, we also address the species ́ reproduction biology.Our study generates reliable sequences of several rarely collected species and furthers understanding on lichen diversity in boreal, boreonemoral and hemiboreal forests especially on dead wood and conifer bark.

Materials and methods
A substantial portion of the sequenced specimens in this study were collected from southern and central Finland as part of a research project that investigated lichen diversity on dead wood (years 2012-2014, Kantelinen et al. 2022b).Logs and stumps of decaying Picea abies trees in decay stages 2, 3, 4 and 5 were inventoried (following Renvall 1995).
In addition, specimens were obtained from the Czech Republic, Japan, Russian Caucasus, Sweden and Ukraine.These specimens were collected from dead wood and bark and sequenced when possible.Geo-coordinates are given in the format WGS84.Relevant specimens were also looked for amongst fresh Micarea collections from Australia, Brazil, Kenya, Rwanda and Tasmania with no success.Herbarium collections and type specimens from the herbaria FR, H, PRA, RBGE, S and UPS were studied.

Species identification
Specimens were identified with a dissecting (Leica S4E) and compound (Leica DM750) microscopes.Anatomical characters and ascospore dimensions were measured in water and K.The number of measured ascospores and conidia depended on their availability, but usually 10-30 were measured and the rest were examined superficially to ensure that they fell into the same size category.To detect and determine the insoluble pigments present in the specimens chemical spot tests with 10% potassium hydroxide (K) and sodium hypochlorite (C) and nitric acid (HNO 3 ) were used (Orange et al. 2010).Fresh material was often not sufficient for thin-layer chromatography (TLC).The specimens are generally quite small and have no or thin thallus, meaning that a substantial part of the collection would need to be taken for TLC.No secondary substances have previously been detected in M. melaeniza, M. nigella or M. osloensis, nor in the similar-looking species M. anterior, M. misella and M. substipitata Palice & Vondrák (Coppins 1983;Czarnota 2007;Vondrák et al. 2022).Thus, TLC is of limited practical value in the study of this species group.
Total genomic DNA was extracted from lichen structures (apothecia, pycnidia and/or thallus) in labs in Finland and the Czech Republic.In Finland, extractions were conducted using DNeasy® Blood & Tissue kit by Qiagen following the manufacturer´s instructions with the following exceptions.Lichen structures of approximately 0.5-1 mm in diam.were ground with mini-pestles in 40 µl of lysis buffer, after which 140 µl of the buffer was added by simultaneously flushing lichen fragments from the mini-pestle.The extracted DNA was eluted in 50 µl of the eluation buffer.Samples were incubated for 5 minutes and centrifuged.After the first elution, a second elution was performed to increase sample availability by adding another 50 µl of the elution buffer, incubated for 5 minutes and centrifuged.The two elutions were stored in the freezer in separate microcentrifuge tubes.In the Czech Republic, extractions were conducted using ISOLATE II DNA Plant Kit (Bioline) according to the manufacturer´s protocol using a cetyltrimethylammonium bromide (CTAB)-based protocol (Aras and Cansaran 2006).
In Finland, PCR reactions were prepared using PuReTaq Ready-To-Go PCR beads (GE Healthcare).The 25 µl reaction volume contained 19 µl dH2O, 0.4 µM of each primer and 4 µL extracted DNA.The ITS and mtSSU regions were used for species identification.PCR was run under the following conditions: initial denaturation for 10 min at 95 °C followed by six cycles of 1 min at 95 °C (denaturation), 1 min at 62 °C (annealing), and 1 min 45 s at 72 °C (extension); for the remaining 35 cycles, the annealing temperature was decreased to 56 °C; the PCR program ended with a final extension of 10 min at 72 °C.The primers used were ITS1LM and ITS2KL (Myllys et al. 1999), andmrSSU1 andmrSSU3R (Zoller et al. 1999) and they were used for PCR amplification and sequencing.In the Czech Republic, PCR reactions for Malíček´s specimens were prepared following the protocol in Malíček et al. (2023).For Vondrák´s and Palice´s specimens PCR reactions were prepared as follows: Polymerase chain reactions were performed in a reaction mixture containing 2.5 mmol/l MgCl2, 0.2 mmol/l of each dNTP, 0.3 µmol/l of each primer, 0.5 U Tag polymerase (TOP-Bio, Praha, Czech Republic) in the manufacturer's reaction buffer, and sterile water to make up a final volume of 10 µl.The ITS and mtSSU regions were used for species identification.PCR was run under the following conditions: for ITS initial denaturation for 3 min at 94 °C followed by 30 cycles of 30 sec at 94 °C (denaturation), 30 sec at 56 °C (annealing), and 2 min at 72 °C (extension); the PCR program ended with a final extension of 7 min at 72 °C.For mtSSU initial denaturation for 10 min at 94 °C followed by 40 cycles of 30 sec at 94 °C (denaturation), 30 sec at 58 °C (annealing), and 2 min at 72 °C (extension); the PCR program ended with a final extension of 7 min at 72 °C.The primers used were ITS1F (White et al. 1990) and ITS4 (Gardes and Bruns 1993), and mrSSU1 (Zoller et al. 1999) and mrSSU7 (Zhou and Stanosz 2001), and they were used for PCR amplification and sequencing.

Phylogenetic analyses
To examine the phylogenetic position of our study species within Micarea s. lat., we ran a preliminary analysis of an mtSSU data matrix using Psora decipiens (Hedw.)Hoffm.from the family Psoraceae as an outgroup, based on the studies by Andersen and Ekman (2005)  The final phylogenies, including 29 newly generated mtSSU and 25 ITS sequences (Table 1), were first aligned with MUSCLE v.3.8.31 (Edgar 2004) using the European Molecular Biology Laboratory, European Bioinformatics Institute's (EM-BL-EBI) freely available web server (http://www.ebi.ac.uk/Tools/msa/muscle/).Based on our previous studies (Launis et al. 2019 a, b) and our preliminary phylogenetic reconstruction of the genus, M. byssacea and M. prasina belonging to Micarea s. str.were selected as outgroups.We ran separate single-marker analyses by using MrBayes3.2.7a and did not detect conflicting clades between the analyses, although missing data was higher in the ITS matrix, and hence decided to concatenate the data by using Mesquite v. 3.61 (Maddison and Maddison 2023).The two-locus data matrix from sequences of 43 specimens included 1245 aligned nucleotide characters, with 707 positions in the mtSSU and 537 positions in the ITS regions.The hypervariable region at the end of the mtSSU was removed from the analyses.Micarea doliiformis is represented by sequences from two specimens that were combined as one, the other specimen represented by an mtSSU sequence and another by ITS sequence (see Table 1).The data matrix was subjected to Bayesian inference using MrBayes (v.3.2.7a)(Ronquist and Huelsenbeck 2003) and to maximum likelihood (ML) analysis using freely available IQ-Tree 1.6.12(Trifinopoulos et al. 2016) web server (http://iqtree.cibiv.univie.ac.at/).For the Bayesian analysis, substitution models were selected by having the MCMC procedure sample across models (Huelsenbeck et al. 2004).The convergence of the four parallel runs was checked after 2000000 generations using Tracer (v.1.5) (Rambaut et al. 2018) and graphed using FigTree (v. 1.4.4).For the ML analysis, model TIM2+F+I+G4 was chosen by having IQ-Tree run the best-fitting substitution model for our one partition matrix, and branch lengths were assumed to be proportional across subsets.Node support was estimated with 1000 bootstrap replicates using the ultrafast bootstrap algorithm.The alignment is available from the Dryad Digital Repository https://doi.org/10.5061/dryad.79cnp5j44.

Results
We present the first molecular phylogeny of M. melaeniza and similar-looking species.We recorded 72 specimens of the M. melaeniza group of which 26 were sequenced successfully.In addition, we downloaded sequences from GenBank (Table 1).The topologies of the Bayesian and ML analyses showed no conflict and hence only the Bayesian tree is shown (Fig. 2).The phylogenies are well-supported and resolved into 18 taxa.Micarea misella, M. globulosella, M. eximia and M. botryoides form basal nodes for the phylogeny.The remaining taxa resolve into four clades that include the M. melaeniza group and its sister groups.The M. melaeniza group, delimited in this study, includes six monophyletic taxa, i.e.M. deminuta, M. melaeniza, M. nigella, M. osloensis and two undescribed species, each represented by two specimens.We describe the first as M. eurasiatica Kantelinen & G. Thor, sp.nov., but refrain from describing the other because of insufficient material and sequence data ("Kantelinen 2640 and Kantelinen 1870"; sequences from the latter specimen are not of sufficient quality and therefore not included in the final analyses even though it formed a monophyletic clade in preliminary analysis).Based on our results the similar looking M. anterior, M. contexta and M. subtipitata are not included in the M. melaeniza group.Instead, they form a sister clade to this group, but the relationship receives no support and is based on limited taxon sampling.Several outgroups, including M. incrassata, M. doliiformis, and Lecania cyrtella have been tested but the overall topology of the phylogeny has not changed.The main distinguishing morphological characters are presented in the species synopsis Table 2. Pigments are presented in Table 3.
In addition to our specimens, we downloaded M. melaeniza and M. nigella sequences from Genbank.Based on our phylogenetic analyses, all of them fall inside Micarea osloensis (OQ646318, OQ646320, OQ717948, OQ646315, OQ717944, OQ646316, in addition OQ717947 and OQ646319 are identical with others and excluded from our final analysis because of repetition).Three sequences from Genbank were left out from our analyses (AY756488, AY756484, OQ717944), because they are substantially different compared to the other sequences in the M. melaeniza group and hence the alignment and phylogenetic analysis became unreliable.Based on blast searches AY756484 is a species of Lepraria, AY756488 perhaps Micarea melaena or M. nitschkeana, and OQ717944 an uncultured fungus.
Our data include a taxon with a high morphological resemblance to Micarea osloensis, a species found only twice before in years 1874 and 2007.Unfortunately, we were not successful in sequencing the old collections.Despite the high morphological resemblance, our new specimens have some subtle differences compared to the type.The type specimen of M. osloensis is a fertile specimen with apothecia, whereas our specimens are usually asexual with pycnidia.The specimens are mostly dimorphic, meaning that the existing sequenced specimens are usually either sexual or asexual, and rarely both.In addition, some of the fresh material is with Cinereorufa green pigment which is not present in the type.See taxonomy section for further info.
Chemistry.Material not sufficient for TLC.
Crystalline granules not present in apothecia or pycnidia.Habitat and distribution.M. eurasiatica is currently known from Finland and Japan.In Finland, the species was collected in a shaded and dense, Picea abies dominated managed forest.In Japan, the species occurred in a semiopen forest with mainly deciduous trees.On both occasions, the species was found growing on dead wood.
Notes.M. eurasiatica is currently known from two collections.The type collection has abundant mesopycnidia, and additionally few small apothecia.

Micarea melaeniza
Mesopycnidia always present, usually numerous, black, sessile or more usually stalked and then 80-300 µm tall, 40-70 µm in diam., stalks simple or branched from the base bearing up to four pycnidia, upper part of the walls greenish-brown and lower part reddish-brown, K+ dull green (the greenish pigment) or sometimes K-.Mesoconidia ellipsoid to short cylindrical 2.5-3.5 × 1.2-1.8µm.Micro-or macropycnidia not seen.
Crystalline granules not present in apothecia, pycnidia or thallus.Chemistry no substances detected by TLC (information based on Coppins 1983 andCzarnota 2007).
Typification.In his original description of Micarea melaeniza, Hedlund (1892) cited material that he had collected in Järvsö in Hälsingland, but without giving further specimen data.There are five specimens of M. melaeniza in S, LD and UPS collected by Hedlund in Järvsö in August 1891 and which all are likely to be part of the original material.Coppins (1983) cited a 'holotype' in S, which constitutes a lectotypification following ICN Art.9.10.There is, however, an additional specimen in S (S L1472) with the same label data, and as Coppins (1983) did not indicate which of these specimens he considered to be the holotype, the lectotypification effectively concerns both specimens.We therefore further specify this by here designating the specimen S L1471 as the lectotype.This specimen was likely the one referred to as holotype by Coppins, as annotation slips from him are included in the envelope.It should be noted that all five type specimens of M. melaeniza are homogeneous.
Habitat and distribution.M. melaeniza occurs on lignum of conifer stumps and logs.Based on sequenced specimens and type, the species is currently known from the Czech Republic, Finland, Sweden, Ukraine and the Russian Caucasus.In addition, M. melaeniza has been reported from Alaska (Spribille et al. 2020), Austria (Berger and Türk 1991, this study) and Mongolia (Palka and Śliwa 2006).Further, it might have been reported as M. nigella and could be found after revising specimens.
Notes.In his monograph of European Micarea species, Coppins (1983) accepted M. melaeniza, and in his interpretation, the species is characterized by having a hymenium with green pigmentation, a dark brown hypothecium without any reaction with K, and black stalked pycnidia containing comparatively short conidia.In the same work, the new species M. nigella was described, which should differ from M. melaeniza by having a purplish brown, K+ green pigment in the hymenium, hypothecium and pycnidial tissues, and slightly larger mesoconidia (M.melaeniza: 2.3-3.6 × 1-1.3 µm vs. M. nigella: 3.4-4.3× 1.2-1.6 µm; Coppins 1983).Czarnota (2007) noted that the amount of purple, K+ green pigment varied considerably in Polish collections determined as M. nigella, and suggested that M. melaeniza and M. nigella could be conspecific.He further noted that Hedlund's original description could be interpreted as indicating the presence of another pigment, the purple, K+ purple pigment and suggested that the differences between Hedlund's and Coppins' descriptions could be due to the studied material having aged (Czarnota 2007).
In our interpretation, M. melaeniza is a species with mostly two pigments: (i) a blackish-green, usually K+ green intensifying pigment, mostly located to the epihymenium but sometimes also in the hymenium and the upper part of the hypothecium (Cinereorufa-green) and (ii) a dark brown, K-pigment in the hypothecium (possibly Superba-brown).The description in Coppins (1983) fits our interpretation quite well, except that the specimens have a K+ greenish reaction due to Cinereorufa-green pigment that is not mentioned by Coppins (l.c.) but is, on the other hand, mentioned in the original description of the species by Hedlund (1892) and seen by us in the type specimen.Pigmentation of M. melaeniza may be more complex, however.One specimen from the Czech Republic (ZP32013) shows patchily purplish, K+ dark green pigment that might be the Melaena-red pigment (in apothecia concentrated mainly in hymenium as darker streaks).This specimen was originally downloaded to GenBank as M. nigella, but it is monophyletic with M. melaeniza in our phylogenetic analyses.More sequenced specimens are needed to understand better the pigment profile of M. melaeniza.
We considered the possibility of M. melaeniza and M. nigella being synonymous.A careful study of the type specimens showed morphological differences, e.g. in the size of conidia and pigmentation of apothecia and pycnidia.A brown or purple-brown, K+ green pigment in the hymenium, hypothecium and pycnidia walls (Melaena-red) of M. nigella is an important difference between M. melaeniza and M. nigella, although this is not true for all the studied specimens as was mentioned above.The difference in pigmentation is also visible in nitric acid, i.e. in M. melaeniza the hypothecium is mostly HNO 3 -(rarely HNO 3 intensifying red), and in M. nigella HNO 3 + purple-red.Compared to M. melaeniza, M. nigella also has longer conidia (3.4-4.5 × 1.2-1.6 µm), slightly shorter hymenium (up to 30 µm) and wider paraphyses (up to 3 µm), as also shown by Coppins (1983).
The molecular study supports the distinction of M. melaeniza and M. nigella (Fig. 2).Our sequenced specimens form two monophyletic clades, and these specimens are morphologically similar with the type specimens (except for ZP32013 discussed above).
Crystalline granules not present in apothecia, pycnidia or thallus.Chemistry no substances detected by TLC (information based on Coppins 1983 andCzarnota 2007).
Habitat and distribution.Micarea nigella occurs mainly on lignum of conifer stumps or fallen trunks, sometimes spreading from wood to dead bryophytes.Based on sequenced specimens and the type material, the species is known from the Czech Republic, Denmark (holotype), Great Britain (paratypes), Finland and Sweden.In addition, M. nigella has previously been reported from boreal and temperate forests in north-western, central and eastern Europe (e.g.Czarnota 2007).
Notes.In external appearance, M. nigella resembles M. melaeniza.The differences between these two species are discussed in detail under M. melaeniza and Coppins (1983).The species also resembles M. botryoides, M. misella and M. osloensis.Micarea botryoides is usually not lignicolous and prefers rain-sheltered microhabitats on various substrata, it has slightly taller pycnidia (up to 400 µm) and longer ascospores (8-13(-16) × 2.3-4 µm) that are often septate (e.g.Coppins 1983).Microscopically, M. misella can be distinguished by the olivaceous pigment that reacts violet instead of dull green in K, and by its hyaline hypothecium (Coppins 1983;Czarnota 2007).Micarea osloensis, on the other hand, is usually K-and its pycnidia are shorter.However, our study includes specimens that are difficult to identify by morphological characters, especially between M. melaeniza, M. nigella and M. osloensis.
One of the distinguishing characteristics of M. nigella is the Melaena-red pigment (K+ green, HNO 3 + purple-red) in the hymenium, hypothecium and pycnidia.In the literature, the pigment is described as `purple` (Coppins 1983;Meyer and Printzen 2000;Czarnota 2007).However, based on our study, the pigment is mostly brown, sometimes with a purplish tinge.The holotype of M. nigella has the Melaena-red pigment, that looks brown with a purplish tinge, but of our three sequenced specimens (Fig. 2, Table 1), one (collection Kantelinen 1971) has no purplish tone, whereas the other two collections (Kantelinen 1974(Kantelinen , 1921) ) have easily detected amounts of purple.Interpreting the colouration can be difficult and confusing, but maybe a helpful hint is that the pigment is always K+ green, even if it looks brown in water.According to our study, the K+ green reaction mostly disappears in 30 minutes.
Occasionally, M. nigella also has a third pigment, the Melaenida-red (K+ purple).This pigment was not found in the Finnish specimens but is sometimes seen in the Central European specimens included in this study and mentioned also by Coppins (1983) and Czarnota (2007).The Melaena-red and Melaenida-red pigments can be intermixed and appear in varying concentrations.
Description.Thallus endoxylic or visible as a thin pale greenish-grey to dark green-grey layer on top of substrate.Photobiont micareoid, 4-7 µm in diam.
Crystalline granules not present in apothecia, pycnidia or thallus.
Habitat and distribution.The type of M. osloensis occurs on soil.Another morphologically identical specimen collected in 2007 occurs on bark of decaying trunk (Palice 11684).Our newer specimens occur on bark, dead wood and dead mosses.The type specimen was collected from Norway from a woodland clearing on the site of an old bonfire, and the newer specimens are from the Czech Republic, Finland, Sweden and Ukraine.In the Czech Republic, M. osloensis occurs commonly from middle to montane elevations.It appears to be toxitolerant and is known in areas with higher levels of air pollution in the past (i.e.acidification by acid rain).The typical habitats are bark on bases and roots of Fagus sylvatica, Larix decidua, Picea abies, Pinus sylvestris.It is abundant also on dead wood and dead bark on stumps, fallen trunks and snags.In Finland, M. osloensis is likely relatively common but overlooked in coniferous forests on bark, dead wood and dead mosses.In both countries, M. osloensis is known from managed and old-growth forests.
Notes.The two previously known M. osloensis specimens, including the type, have not been sequenced, although an unsuccessful sequencing attempt of a specimen collected by Palice (11684) was made by Kantelinen in 2011, and therefore we cannot compare our new specimens to the type of M. osloensis using DNA.Subtle morphological features differentiate the type from new specimens, i.e. taller hymenium and asci.Most of the new specimens are Kand have only the Superba-brown pigment, similar to the type.However, some specimens have a K+ greenish, HNO 3 + purple reaction in the epihymenium and pycnidial walls suggesting the presence of the Cinereorufa-green pigment which is not known from the type of M. osloensis.Specimens with the Cinereorufa-green pigmentation appear to be more frequent in the Czech Republic.The Finnish specimens have sometimes olivaceous tones that are K-but slightly HNO 3 + purple.
Another difference between the type of M. osloensis and our newly sequenced specimens is reproduction.The type specimen has apothecia and no pycnidia.The new specimens, on the other hand, often have shortly stipitate pycnidia.Our specimens appear to be dimorphic, however, so that the specimens represent either sexual (rare) or asexual reproduction modes which are monophyletic in DNA level.
Because of overlapping variation in reproduction and pigmentation between the type of M. osloensis and our new specimens, we cannot exclude the possibility that they are conspecific.On the other hand, we also cannot exclude the possibility that the new specimens represent a yet undescribed taxon in the M. melaeniza group.
M. osloensis resembles M. eurasiatica, M. melaeniza, M. misella and M. nigella.The most important characters of M. osloensis are the combination of sessile to shortly stalked pycnidia, mesoconidia of the size 3.5-4.5(-5)× 1.2-1.5 (-1.8) µm, warm-brown, sometimes olivaceous colouration in apothecia our fresh specimens underline this issue, and are discussed in the notes under M. melaeniza, M. nigella and M. osloensis.According to our study, the species in the M. melaeniza group are characterized by having a thin or endosubstratal thallus and dark, sessile to stipitate mesopycnidia that often extrude a white mass of conidia at their top.Apothecia are often absent, but when present, they are 0.1-0.3mm wide and brown to black in colour.They have hyaline or slightly coloured hymenium, dimorphic paraphyses, simple spores and dark hypothecium where hyphae are surrounded by brown pigment giving them an unevenly coloured appearance, also noted by Coppins (1983).Based on results of this study, the size of pycnidia and mesoconidia, and to some extent also the general pigmentation in K and HNO 3 are the most useful morphological characters in separating the species, however in some cases DNA sequencing is the only reliable way for identification.Micarea eurasiatica, and M. olivacea develop sessile to emergent pycnidia, M. osloensis emergent to shortly stipitate pycnidia, and M. melaeniza and M. nigella develop stipitate pycnidia.Micarea deminuta does not develop distinctive stalked pycnidia, at least according to current knowledge.The Cinereorufa-green pigment (K+ green, HNO 3 + purple) is present in pycnidia and apothecia of M. eurasiatica, M. deminuta, M. melaeniza, M. nigella, M. olivacea (according to Coppins 1983) and occasionally in M. osloensis in varying concentration, accompanied by the Superba-brown pigment (K-).The brown or purple-brown Melaena-red pigment (K+ dull sordid green) is present in M. nigella (and possibly in one specimen of M. melaeniza) and is sometimes intermixed with the Melaenida-red pigment (K+ purplish-red) (Table 3).
Our study indicates that the pigmentation of some species may correlate with geography.In the central European specimens some pigments are encountered more often or higher concentrations than in Fennoscandia.For example, in the Fennoscandian specimens of M. osloensis, the Cinereorufa-green pigment is barely visible even when using HNO 3 , and the Melaenida-red pigment in M. nigella was not found at all.In the central European specimens, however, both pigments were found, and sometimes in relatively high concentrations.The central European material also includes samples that appear to be morphologically "intermediate" between species, for example M. melaeniza (ZP32013) may have Melaena-red pigment like M. nigella, although its mesoconidia size and DNA profile is similar to M. melaeniza.Some of the "intermediate" specimens have not been sequenced and therefore we do not know their identity or whether they are undescribed species.Obviously, more work is needed to understand the pigments and species in this group.
Because of these morphological challenges, we even considered that the species in the M. melaeniza group are conspecific, i.e. variation of just one species.However, we excluded this possibility because of several reasons 1) molecular differences, 2) existing morphological differences, even though they may be hard to interpret, 3) the type specimens studied are not morphologically conspecific, 4) the types correspond in morphology to most of our specimens.Based on morphology, one might suggest that M. melaeniza, M. nigella and M. osloensis are variation of one species, but according to our phylogenies, they are not monophyletic without M. deminuta, M. eurasiatica and Micarea sp.Even between M. melaeniza and M. nigella, the pigmentation is mostly different (Superba brown vs. Melaena-red), and although these pigments may look quite similar to the human eye, they may have different ontologies and evolutionary paths.
According to morphological studies by Coppins (1983), M. botryoides and M. melaeniza are relatives (group G) and possibly close to M. contexta, M. eximia, M. nigella and M. olivacea (group H).Our Bayesian and ML phylogenies conclude that groups G and H are intermixed, however, based on DNA sequencing M. eximia is likely not a close relative of the M. melaeniza group (unpublished data) and molecular relationships of M. olivacea are still unknown.Our phylogenetic analyses also show that M. melaeniza and M. osloensis are sisters, a relationship that has not been noted in previous publications, but the latter clade is not supported (0.68 / 74).A closer look at the sequences and alignment shows that there are nucleotide differences in the mtSSU region between M. melaeniza and M. osloensis (ca.1-2% between specimens Kantelinen 2430 and 1923), but the ITS regions are nearly identical with only two nucleotide differences.At least three conclusions could be drawn from these results.1) The two clades are, in fact, two species as we suggest in our study.This conclusion is supported by molecular and morphological data, to some extent at least, 2) The two clades are conspecific and represent morphological and molecular variation of M. melaeniza, 3) Our new specimens of M. osloensis are not conspecific to the type of M. osloensis or M. melaeniza, but instead a scientifically undescribed species.Any conclusion we make suffers from the uncertainty caused by the M. melaeniza and M. osloensis type specimens not having sequenced, which means that we cannot compare our sequences to the types and the connections between fresh and old specimens are based solely on morphology.This is also the reason why we refrain from describing our new specimens of M. osloensis as a new species to science.The type specimens are over 100 years old and hence likely beyond successful DNA sequencingbased on our experience, sequences are usually difficult to get from Micarea specimens just over 3 years old, and nearly impossible when over 6 years old.
All species in the M. melaeniza group are either obligate or facultative lignicoles.According to a previous study, the wood-inhabiting lifestyle of Micarea species influences their reproductive biology: obligate lignicoles primarily reproduce asexually, likely due to the transient nature of decaying wood, which imposes a time constraint on the species occupying it (Kantelinen et al. 2022a).Asexual reproduction via mesoconidia is likely a more rapid and efficient strategy than sexual reproduction.In the here studied M. melaeniza group and relatives, several species are found mostly asexual, viz.M. melaeniza, M. nigella, M. osloensis, M. eurasiatica, M. anterior and M. substipitata.
Based on our field experience as well as previous works by Coppins (1983) and Czarnota (2007), the species in the M. melaeniza group are probably common in boreal and hemiboreal forests, both in natural and managed forests.In spite of this, their small size and often anamorphic lifestyle make them easily overlooked, resulting in rare mentions in ecological studies or species inventories.We hope that the morphological and molecular features presented in this study will pave the way for future research endeavors.

Figure 1 .
Figure 1.Typical habitats for species in the M. melaeniza group in boreal and boreonemoral forests a Koli National Park, Eastern Finland (photo: Kantelinen) b Nikko National Park, Central Honshu, Japan near the type locality of M. eurasiatica sp.nov.(photo: Thor).

Figure 4 .
Figure 4. Morphological and anatomical features of old and new Micarea osloensis collections A, B old Micarea osloensis A habit, apothecia on soil (Holotype) B apothecial section in water (Palice 11684, H) C-E new Micarea osloensis (Kantelinen 2648, H) C mesopycnidia extruding mesoconidia as a white drop D apothecial section in water E mesopycnidia on dead wood and mosses.Scale bars: Habit 0.5 mm (A, C, E); Apothecial sections 100 µm (B, D).

Table 1 .
List of Micarea specimens used in the phylogenetic analysis with locality, voucher information and GenBank accession numbers.